Volume 12, Issue 5, Pages 495-503 (May 2000) CCR6 Mediates Dendritic Cell Localization, Lymphocyte Homeostasis, and Immune Responses in Mucosal Tissue  Donald N Cook, Dina M Prosser, Reinhold Forster, Jiwen Zhang, Nelly A Kuklin, Susan J Abbondanzo, Xiao-Da Niu, Shu-Cheng Chen, Denise J Manfra, Maria T Wiekowski, Lee M Sullivan, Sidney R Smith, Harry B Greenberg, Satwant K Narula, Martin Lipp, Sergio A Lira  Immunity  Volume 12, Issue 5, Pages 495-503 (May 2000) DOI: 10.1016/S1074-7613(00)80201-0

Figure 1 Analysis of CCR6 and MIP-3α Expression in the Mouse (A) Northern RNA blot of MIP-3α (top panel) and CCR6 (bottom panel) mRNAs. Abbreviations: ad, adrenal; PP, Peyer's patch; ut, uterus; st, stomach; ov, ovary; lu, lung; br, brain; th, thymus; k, kidney; m, skeletal muscle; sp, spleen; h, heart; s.i., small intestine; l.i., large intestine; te, testes; bm, bone marrow; sc, spinal cord; li, liver; ln, lymph nodes. (B–E) In situ hybridization of Peyer's patch with an antisense probe corresponding to MIP-3α (B and C) and CCR6 (D and E). Magnification: (B) and (D), 10×; (C) and (E), 60×. No specific signal was seen with control (sense) probes. Labeled are PP, Peyer's patch; V, villi; SED, subepithelial dome; Ep, epithelium. Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)

Figure 2 CCR6 Gene Targeting (A) The targeting construct. The neomycin resistance gene (neo) is flanked by a 5.5 kb and a 1.1 kb DNA fragments from the CCR6 locus. (B) CCR6 locus. Restriction sites shown are HindIII (H), BamHI (B), and NotI (N). Sizes are in kilobases. The single CCR6 exon is shown by a black rectangle and an arrow indicates the direction of transcription. (C) Structure of the targeted locus. The sizes of the expected fragments are shown in kilobases. Arrowheads represent the primers used in a polymerase chain reaction (PCR) to identify targeted ES cell clones. The probe used for Southern blot confirmation of targeted clones is shown. (D) Analysis of MIP-3α binding. Radiolabeled MIP-3α was incubated with membranes of splenocytes from wild-type and CCR6−/− mice. Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)

Figure 3 Confocal Analysis of Cell Populations in Peyer's Patch The antibodies used are as follows: (A and B) CD11b (green) and CD11c (red), double-positive cells stain yellow; (C and D) IgD (green), DEC 205 (red), and Thy-1 (blue); (E and F) IgD (green), IgM (red), and Thy-1 (blue). Wild-type mice, (A, C, E); CCR6−/− mice, (B, D, F). Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)

Figure 4 Flow Cytometric Analysis of T Lymphocytes in Intestinal Mucosa The absolute numbers of the indicated lymphocyte subsets are shown for intestinal epithelium (A) and lamina propria (B). Data shown are representative of a minimum of three experiments. p values (two-tailed t test) for all cell populations shown are less than 0.05 except for TCRγδ cells and B220 cells. Wild-type mice, closed rectangles; CCR6−/− mice, open rectangles. (C) Immunohistologic staining of CD8β-expressing IEL. Representative longitudinal (top panel) and cross (bottom panel) sections of wild type (+/+) and CCR6−/− mice (−/−) are shown. Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)

Figure 5 Ig in Serum of KLH-Immunized Mice Wild-type mice (closed rectangles) and CCR6−/− mice (open rectangles) were immunized with KLH, and serum Ig levels determined. (A) Total Ig. (B) KLH-specific Ig. An asterisk denotes differences between wild-type and CCR6−/− mice in which p < 0.05. Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)

Figure 6 Rotavirus Infection Mice were infected with 1 × 105 50% diarrhea dose (DD50) of murine rotavirus strain EC and assayed for level of intestinal anti-rotavirus IgA (A), anti-rotavirus serum IgG (B), and rotavirus (C) antigen in stools as previously described (Rosé et al. 1998). Data shown for IgA and IgG is from one of two representative experiments (n = 8/group) having similar results. For antigen, data shown represents pooled results (n = 16/group) of two experiments. Wild-type mice, closed dots and rectangles; CCR6−/− mice, open dots and rectangles. Immunity 2000 12, 495-503DOI: (10.1016/S1074-7613(00)80201-0)